Abstract Aneuploidy,thestateinwhichcellscarryanincorrectnumberofchromosomes,isahallmarkofhuman cancers.Over85%ofcancersareaneuploid,andhigherratesofaneuploidyareoftenassociatedwithpoor patientprognosis.Aneuploidtumorscandisplayheterogeneouskaryotypes,whichunderlieheterogeneityin cellularphenotypes.Thispresentsaspecificchallengeintreatinganeuploidtumors,becausetheycanrapidly evolvemechanismstoevadetreatment.Giventhehighincidenceofaneuploidyindiversecancertypes,an attractivestrategywouldbetoselectivelysensitizecellstotheaneuploidstateitself,especiallyincombination withchemotherapydrugs.Thishasbeenchallenging,inlargepartbecauseitremainsunclearhowaneuploid cancercellscantolerateextraDNAcontentinthefirstplace.Wehavetakenanovelperspectivetothis challenge,bystudyingwildstrainsofbuddingyeastSaccharomycescerevisiaethatarenaturallytolerantto extrachromosomes.Yeastisapowerfulmodelfordissectingcellularbiology,becausemanyofthe mechanismsanddefensestrategiesareconservedinhumans.Bycomparinganeuploidy-tolerantwildstrains toawell-studiedlaboratorystrainthatisunusuallysensitivetoaneuploidy,wediscoveredasinglegenethat, whendeleted,produceslittletonophenotypeineuploidstrains,butrenderscellsverysensitivetoextra chromosomes.Thus,wecansensitizecellstoaneuploidywithoutproducingmajorphenotypesinthenormal euploidcells.Thegene?Ssd1?hasbeenimplicatedinmRNAlocalization,translationalcontrol,and chromosomemaintenanceamongotherthings,butthemechanismsremainunclear.Webelievethatthe processofaneuploidytoleranceisconservedbetweenyeastandhumans.ThehumanorthologofSsd1, hDis3L2,sharesseveralfeatureswithSsd1,includinglinkstotranslationalregulation,P-bodylocalization,and properchromosomesegregation.Thegoalofthisproposalistwofold:1)toidentifythemechanismthrough whichSSD1deletionsensitizesyeasttoaneuploidyand2)tousethisinformationtotestifknockdownof orthologousfunctionssensitizescancercelllinestoaneuploidy,withandwithoutchemotherapytreatment. Aim1willusegenomics,proteomics,single-moleculeRNAfluorescenceinsituhybridization(FISH),and singe,live-cellimagingtotesttheroleofSsd1inaneuploidytolerance.Aim2willleveragetheseinsightsto testiforthologousmechanisms,includingknockdownofthehumanorthologhDis3L2,cansensitizebreastand coloncancercelllinestoaneuploidy,withandwithoutpaclitaxeltreatment.Thisaimwilluseapowerful systemtoproduceisogenicsetsofeuploidandaneuploidhumancells,enablingsensitivedissectionof phenotypesthatarespecifictotheaneuploidstate.Resultsofthisworkwillexpandourunderstandingofthe functionofSsd1/hDis3L2andcouldpavethewaytonewtherapeuticapproachestotargetaneuploidcells.